Cells actively regulate the macromolecular excluded volume of the cytoplasm to maintain the reciprocal fraction of free aqueous solution that is optimal for intracellular processes. However, the mechanisms whereby cells sense this critical parameter remain unclear. The mechanosensitive channel of small conductance (MscS channel), which is the major regulator of turgor in bacteria, mediates efflux of small osmolytes in response to increased membrane tension. At moderate sustained tensions produced by a decrease in external osmolarity, MscS undergoes slow adaptive inactivation; however, it inactivates abruptly in the presence of cytoplasmic crowding agents. To understand the mechanism underlying this rapid inactivation, we combined extrapolated and equilibrium molecular dynamics simulations with electrophysiological analyses of MscS mutants to explore possible transitions of MscS and generated models of the resting and inactivated states. Our models suggest that the coupling of the gate formed by TM3 helices to the peripheral TM1–TM2 pairs depends on the axial position of the core TM3 barrel relative to the TM1–TM2 shaft and the state of the associated hollow cytoplasmic domain (“cage”). They also indicate that the tension-driven inactivation transition separates the gate from the peripheral helices and promotes kinks in TM3s at G113 and that this conformation is stabilized by association of the TM3b segment with the β domain of the cage. We found that mutations destabilizing the TM3b–β interactions preclude inactivation and make the channel insensitive to crowding agents and voltage; mutations that strengthen this association result in a stable closed state and silent inactivation. Steered simulations showed that pressure exerted on the cage bottom in the inactivated state reduces the volume of the cage in the cytoplasm and at the same time increases the footprint of the transmembrane domain in the membrane, implying coupled sensitivity to both membrane tension and crowding pressure. The cage, therefore, provides feedback on the increasing crowding that disengages the gate and prevents excessive draining and condensation of the cytoplasm. We discuss the structural mechanics of cells surrounded by an elastic cell wall where this MscS-specific feedback mechanism may be necessary.

The mechanosensitive channel of small conductance (MscS) is a bacterial tension-driven osmolyte release valve with homologues in many walled eukaryotic organisms. When stimulated by steps of tension in excised patches, Escherichia coli MscS exhibits transient opening followed by reversible adaptation and then complete inactivation. Here, we study properties of the inactivation transition, which renders MscS nonconductive and tension insensitive. Using special pressure protocols we demonstrate that adaptation and inactivation are sequential processes with opposite tension dependencies. In contrast to many eukaryotic channels, which inactivate from the open state, MscS inactivates primarily from the closed state because full openings by preconditioning pulses do not influence the degree of inactivation, and saturating tensions keeping channels open prevent inactivation. The easily opened A98S mutant lacks inactivation completely, whereas the L111S mutant with a right-shifted activation curve inactivates silently before reaching the threshold for opening. This suggests that opening and inactivation are two independent tension-driven pathways, both starting from the closed state. Analysis of tension dependencies for inactivation and recovery rates estimated the in-plane expansion (ΔA) associated with inactivation as 8.5 nm 2 , which is about half of the area change for opening. Given that the interhelical contact between the outer TM1–TM2 pairs and the core TM3s is the force-transmitting path from the periphery to the gate, the determined ΔA now can be used as a constraining parameter for the models of the inactivated state in which the lipid-facing TM1–TM2 pairs are displaced and uncoupled from the gate.

Mechanosensitive channel of small conductance (MscS), a tension-driven osmolyte release valve residing in the inner membrane of Escherichia coli , exhibits a complex adaptive behavior, whereas its functional counterpart, mechanosensitive channel of large conductance (MscL), was generally considered nonadaptive. In this study, we show that both channels exhibit similar adaptation in excised patches, a process that is completely separable from inactivation prominent only in MscS. When a membrane patch is held under constant pressure, adaptation of both channels is manifested as a reversible current decline. Their dose–response curves recorded with 1–10-s ramps of pressure are shifted toward higher tension relative to the curves measured with series of pulses, indicating decreased tension sensitivity. Prolonged exposure of excised patches to subthreshold tensions further shifts activation curves for both MscS and MscL toward higher tension with similar magnitude and time course. Whole spheroplast MscS recordings performed with simultaneous imaging reveal activation curves with a midpoint tension of 7.8 mN/m and the slope corresponding to ∼15-nm 2 in-plane expansion. Inactivation was retained in whole spheroplast mode, but no adaptation was observed. Similarly, whole spheroplast recordings of MscL (V23T mutant) indicated no adaptation, which was present in excised patches. MscS activities tried in spheroplast-attached mode showed no adaptation when the spheroplasts were intact, but permeabilized spheroplasts showed delayed adaptation, suggesting that the presence of membrane breaks or edges causes adaptation. We interpret this in the framework of the mechanics of the bilayer couple linking adaptation of channels in excised patches to the relaxation of the inner leaflet that is not in contact with the glass pipette. Relaxation of one leaflet results in asymmetric redistribution of tension in the bilayer that is less favorable for channel opening.

Mechanosensitive channels of small conductance (MscS) are ubiquitous turgor pressure regulators found in many walled cells and some intracellular organelles. Escherichia coli MscS acting as a tension-activated osmolyte release valve shows a nonsaturable conductance (1.2 nS in a 39 mS/cm electrolyte) and weak preference for anions. Pursuing the transition pathways in this channel, we applied the extrapolated motion protocol (cycles of displacements, minimizations, and short simulations) to the previously generated compact resting conformation of MscS. We observed tilting and straightening of the kinked pore-forming TM3 helices during the barrel expansion. Extended all-atom simulations confirmed the stability of the open conformation in the bilayer. A 53° spontaneous axial rotation of TM3s observed after equilibration increased the width and polarity of the pore allowing for stable voltage-independent hydration and presence of both cations and anions throughout the pore. The resultant open state, characterized by a pore 1.6 nm wide, satisfied the experimental conductance and in-plane expansion. Applied transmembrane electric field (±100 to ±200 mV) in simulations produced a flow of both K + and Cl − , with Cl − current dominating at higher voltages. Electroosmotic water flux strongly correlated with the chloride current (∼8 waters per Cl − ). The selectivity and rectification were in agreement with the experimental measurements performed in the same range of voltages. Among the charged residues surrounding the pore, only K169 was found to contribute noticeably in the rectification. We conclude that (a) the barrel expansion involving tilting, straightening, and rotation of TM3s provides the geometry and electrostatics that accounts for the conductive properties of the open pore; (b) the observed regimen of ion passage through the pore is similar to electrodiffusion, thus macroscopic estimations closely approximate the experimental and molecular dynamics-simulated conductances; (c) increased interaction of the opposing ionic fluxes at higher voltages may result in selectivities stronger than measured near the reversal potential.